UW-Madison researchers have developed a universal botulinum neurotoxin production strain using Clostridium tetani. C. tetani ATCC 454 is an atoxic strain lacking the tetanus toxin gene (tent) as well as its regulator and promotor region (tetR). The researchers used an E. coli conjugative donor strain to move a plasmid expressing a botulinum toxin gene into the tetani strain. By utilizing a combination of antibiotic treatments, they could select the C. tetani organisms containing the expression vector. The researchers also designed the expression vector to contain an optimal origin of replication, a strong promoter, and they had to determine the optimal media in which to grow the transfected organism. They found that they could express a mutated form of botulinum toxin A1 gene that produces a toxin with very low toxicity using the Clostridium tetani without the toxin protein complexing with the stabilizing proteins commonly seen in botulinum toxin fermentation.